Virus-like nanoparticles as a novel delivery tool in gene therapy

Accepted Manuscript Virus-like nanoparticles as a novel delivery tool in gene therapy Jaison Jeevanandam, Kaushik Pal, Michael K. Danquah PII:

S0300-9084(18)30316-X

DOI:

https://doi.org/10.1016/j.biochi.2018.11.001

Reference:

BIOCHI 5539

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Biochimie

Received Date: 26 August 2018 Accepted Date: 1 November 2018

Please cite this article as: J. Jeevanandam, K. Pal, M.K. Danquah, Virus-like nanoparticles as a novel delivery tool in gene therapy, Biochimie, https://doi.org/10.1016/j.biochi.2018.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Abstract.

Viruses are considered as natural nanomaterials as they are in the size range of 20 to 500 nm

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with a genetical material either DNA or RNA, which is surrounded by a protein coat capsid. Recently, the field of virus nanotechnology is gaining significant attention among researchers. Attention is given to the utilization of viruses as a nanomaterial for medical, biotechnology and

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energy applications. Removal of genetic material from the viral capsid creates empty capsid for drug incorporation and coating the capsid protein crystals with antibodies, enzymes or aptamers

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will enhance their targeted drug deliver efficiency. Studies reported that these virus-like nanoparticles have been used in delivering drugs for cancer. It is also used in imaging and sensory application for various diseases. However, there is a hesitation among researchers to utilize virus-like nanoparticles in targeted delivery of genes in gene therapy, as there is a

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possibility of viral mutation. Thus, the current review focuses on highlighting the possibilities of using virus-like nanoparticles for targeted gene delivery. In addition, other biomedical applications that are explored using virus-like nanoparticles and their probable mechanism of

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delivering genes were also discussed.

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Keywords: Virus-like nanoparticles; Gene therapy; Targeted drug delivery; Biomedical application; Natural nanomaterials

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Graphical abstract

ACCEPTED MANUSCRIPT Virus-like nanoparticles as a novel delivery tool in gene therapy Jaison Jeevanandam1, Kaushik Pal2* and Michael K. Danquah3 1

Department of Chemical Engineering, Faculty of Engineering and Science, Curtin University, CDT250 Miri, Sarawak 98009, Malaysia. 2

Bharath Institute of Higher Education and Research, Bharath University, Department of

Nanotechnology, Research Park, 173 Agharam Road, Selaiyur, Chennai 600073, Tamil Nadu (India). 3

Civil & Chemical Engineering Department, University of Tennessee, Chattanooga, TN 37403, Unites

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*Corresponding author: Prof. (Dr.) Kaushik Pal

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States.

Research Professor, (Group Leader & Independent Scientist)

Bharath University, BIHER Research Park, Department of Nanotechnology, Chennai 600073, Tamil Nadu (India).

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E-mail: ([email protected]) ; ([email protected])

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ACCEPTED MANUSCRIPT Abstract Viruses are considered as natural nanomaterials as they are in the size range of 20 to 500 nm with a genetical material either DNA or RNA, which is surrounded by a protein coat capsid. Recently, the field of virus nanotechnology is gaining significant attention among researchers. Attention is given to the utilization of viruses as a nanomaterial for medical, biotechnology and energy applications. Removal of genetic material from the viral capsid creates empty capsid for drug incorporation and

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coating the capsid protein crystals with antibodies, enzymes or aptamers will enhance their targeted drug deliver efficiency. Studies reported that these virus-like nanoparticles have been used in delivering drugs for cancer. It is also used in imaging and sensory application for various diseases. However, there is a hesitation among researchers to utilize virus-like nanoparticles in targeted delivery of genes in gene therapy, as there is a possibility of viral mutation. Thus, the current review focuses on highlighting the

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possibilities of using virus-like nanoparticles for targeted gene delivery. In addition, other biomedical applications that are explored using virus-like nanoparticles and their probable mechanism of delivering genes were also discussed.

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Keywords: Virus-like nanoparticles; Gene therapy; Targeted drug delivery; Biomedical application;

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Natural nanomaterials

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ACCEPTED MANUSCRIPT 1. Introduction The process of transferring a gene that can alter endogenous gene expression or encode a functional protein in a cell is called gene therapy, which is considered as an alternate to enzyme or protein replacement therapy, due to their unique alteration ability of target function [1, 2]. In 1989, the initial gene transfer experiment was performed by infiltrating tumor in lymphocytes, genetical tagging via retrovirus vector and reinforced to cure tumor cells in order to prove the possibility of genetic

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modifications and gene therapy in patients without harming them [3]. Later, it was reported that in 2017, 2597 clinical trials of gene therapies were performed in 38 countries due to the advancements in the gene transfer technology [4]. The therapy can be initiated by identification of mutant or repaired gene, followed by cloning the identical healthy gene called transgene and loading them in a vehicle called vector [5]. Gene therapy can be given to a patient either through viral, non-viral or engineered

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vectors. Viral vectors such as retrovirus, adenovirus, Herpes simplex virus 1 (HSV-1), vaccinia virus, lentivirus, baculo virus, Moloney Murine Leukemia virus (MLV proteins) and adeno-associated virus (AAV) were utilized for transferring genes in conventional gene therapy [6]. In this process, the genetic material in the viruses is replaced with the therapeutic gene that possesses a promoter and a required

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sequence of genetic code to be delivered in target site to suppress, augment or repair disease causing genes [7]. However, major drawbacks such as immunogenicity, cytotoxicity, inflammatory reaction towards viral vectors and insertional mutagenesis of gene delivering viral vectors leads to the upsurge of non-viral vectors for enhanced gene therapy [8]. Naked viral DNA segments also known as viroid, particle and chemical vectors are the types of non-viral vectors that are used to deliver genes by either plasmid (direct), chemical or physical administration. Non-viral vectors have advantages such as efficiency, low cost, specificity, simple production method, gene expression in less duration, reduced

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pathogenicity, less immunotoxicity and are safer than viral vectors. These advantages have increased their usage for gene therapy and caused the entry of numerous non-viral vector products into the clinical trials from the year 2004 to 2013 [9]. However, drawbacks such as less effectual gene transport and unloading of target cells, problems in commercialization, vigorous immune response and regulatory issues are prevalent as challenges for non-viral vectors to be used in efficient gene therapy

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[10-13].

In recent times, engineered vectors have gained applicational importance in gene therapy to overcome the challenges in using viral vectors [14]. Surface modification of viral capsid [15] via genetic

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engineering [7] and chemical methods are used to fabricate engineered viral vectors [16]. However, high efficiency of engineered vectors in gene delivery was not yet achieved due to the chances of an unprecedented reaction between the transgenes and the capsid, and mutation ability of viral capsid [17]. Meanwhile, virus-like nanoparticles are introduced as engineered viral vectors that are extensively useful as carriers in delivering drugs to targeted sites [18]. Very few studies were performed to investigate the ability of virus-like nanoparticles in delivering genes, similar to drugs. Thus, these virus-like nanoparticles are proposed in the current review to be a novel gene therapy vector in future for delivering therapeutic genes in the target site. Further, the recent advancements in the fabrication of these nanoparticles, which leads to various biomedical and pharmaceutical applications are listed and possible mechanism of virus-like nanoparticles as vectors in gene therapy were also discussed. 2. Viruses – a bridge between living organisms and non-living materials Viruses are the simplest forms of microorganisms which generally are composed of genetic material either DNA or RNA and a symmetrical protein capsid [19]. Certain viruses such as T4 bacteriophage

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ACCEPTED MANUSCRIPT possess short and long tail fibers of proteins which is responsible for sensing and creating infection in host cells [20]. A complete virus particle is called as virion and its main function is to deliver its genetic material (genome) into the host cell through transcription [21]. Even though, they contain genetic material and cause diseases in host cells, viruses were not considered as a living organism, when present outside the cell, even by the International Committee on Taxonomy of Viruses and some virologists [22, 23]. Inactiveness outside a host organism, absence of cytoplasm and cell organelles lead to the conclusion that viruses are an exceptional organism to be named as a living organism [24].

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In the aspect of evolution, viruses do not have common ancestors (phylogeny) and are grouped under polyphyletic as they are not delineated with self-centered genetic elements called plasmids [25]. Thus, they are commonly called the bridge between living organism and non-living materials [26]. Outside host cells, viruses are metastable molecules with assemble and disassemble potential for delivering genetic material into the host cells [27]. Viruses are also unique as they lack structural continuity [28],

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capability of rapid evolution and recombination compared to cells [29], and adapt with the gene of hosts (gene robbers) for their survival [30]. The recent developments in imaging viruses and their life cycle, such as electron and in situ microscopes lead to the advancements in exploring their nature and properties for utilizing them in biomedical applications [31].

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Generally, viruses are categorized into virus nanoparticles, which contain capsid and specific genomes [32] whereas virus-like nanoparticles are the empty protein capsid [33]. There are more than 30,000 viruses that are grouped into 71 families, 164 genera and 3600 species [34]. Further, viruses are classified based on the host cells in which they cause infection such as plants [35], bacteriophages [36], animal and human cells [37]. It is noteworthy that the viruses that cause infection in plants do not possess the ability to infiltrate infection in humans and animals and vice versa [38]. Among these wide range of viruses, only plant viruses and certain bacteriophages and animal viruses are utilized to form

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virus-like nanoparticles for medical, therapeutic and pharmaceutical applications as there is a lesser probability of inhibiting chemical reaction and mutation possibility in human cells [39]. 3. Virus-like nanoparticles

The capsid of viruses without genetic material, that are made up of proteins named as virus-like nanoparticles, are rigid and firm structures that can withhold drug molecules in a confined nanosized

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spatial distribution [40]. These are well-defined protein based geometries at the atomic level with attractive self-assembly properties that are alterable by gene manipulation and exceptional biocompatibility [41]. Usually, virus-like nanoparticles are a cluster of two or fewer protein molecules

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that are currently under extensive research for reagent, scaffold and catalytic applications for chemical reactions [42]. The void space in the viral capsid after the removal of genetic material is used as an excellent nanocarrier due to their distinct morphologies with uniformity, easy functionalization and varied sizes ranging from 10 nm to a few microns [43]. Figure 1 shows the viral capsid of several viruses that are used as virus-like nanoparticles. Mostly, capsids of plant viruses are used as nanocarriers as their genomes are different from animal and human viruses to avoid any unexpected genetic mutations [44]. In recent times, viral capsids from viruses such as cowpea chlorotic mottle virus (CCMV) [45], cowpea mosaic virus (CPMV) [46], red clover necrotic mosaic virus (RCNMV) [47], MS2 RNA-containing bacteriophage [48], Qβ bacteriophage [49], M13 bacteriophage [50], tobacco mosaic virus (TMV) [51], turnip yellow mosaic virus (TYMV) [52], tomato bushy stunt virus [53], brome mosaic virus (BMV) [54], the Canine parvovirus (CPV) [55], the Turnip crinkle virus [56] and flock house virus (FHV) [57] were used as nanocarriers to entrap drug molecules.

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Figure 1: Viral capsids from different viruses that are used as virus-like nanoparticles [58, 59]; Accession number: HBV – 2QIJ, PV – 3IYJ, HEV – 2ZTN, AIMV – AMV, FHV – 2Q26, MS2 – 2MS2, Qbeta – 1QBE, CPMV – 1NY7.

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The virus-like nanoparticles are fabricated by methods such as genetic engineering [60], bioconjugate chemistry [61], infusion [62], mineralization [63] and self-assembly [64]. The genetic code of the virus determines the proteins to be present in the viral capsid [65]. Genetic engineering and Bioinformatics are used to alter the viral genome, which further helps to modify their protein pattern in capsid [66, 67]. Transgenesis of genes from one organism to another or modifying mutated gene (responsible for

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disease) with healthy gene is possible via genetic engineering processes [68] and recent developments in synthetic genomics lead to insertion of various novel synthetic amino acids into the viral genome [33], short peptide sequence [33] and moieties in capsid for targeting receptors to be used in

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pharmaceutical applications [69]. Insertion of whole protein, protein domains and creating hybrid viruses with distinct protein expression in viral capsids also have been achieved in recent times [70, 71]. Currently, CRISPR genetic sequences are widely used in the genetic engineering of viruses to be utilized as virus-like nanoparticles [72]. Similarly, bioconjugation is another alternate method to target and modify both natural and synthetic amino acids on virus capsid, which is not achievable through genetic engineering [73]. Chemical conjugations via functional groups such as cysteine, lysine and aspartic/glutamic acid are conventionally used to alter viral genomes [74]. Recently, conjugation of viruses using biomolecules, especially enzymatic and antigens, is gaining much applicational importance for altering viruses as useful nanoparticles [73, 75]. Infusion is the method of removing the viral genome and the insertion of foreign material like drugs to deliver them at the target site [76]. This method is highly useful for the fabrication of virus-like nanoparticles [77]. The pores present in the viral capsids are used to diffuse the drug molecules and electrostatic or affinity mediated interactions with the proteins in viral capsid from the inside helps in

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ACCEPTED MANUSCRIPT the retention of those molecules [78]. Infusion of drugs for controlling the target delivery and dyes for imaging are the current trend in fabricating virus-like nanoparticles for biomedical applications [79]. Likewise, biomineralization also serves as templates for the formation of virus-like nanoparticles with desired size and morphology. Mineralization-directing peptides and fine-tuned electrostatics are the biomineralization techniques that are extensively utilized for the fabrication of interior and exterior surfaces of virus-like nanoparticles, specifically with rod and icosahedral morphologies [80, 81]. Recently, nanoparticle encapsulated multifunctional virus-like nanoparticles are fabricated via a

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biomineralization process [82, 83]. Self-assembly is a unique method of assembling viral proteins to form an encapsulation of nanoparticles such as gold nanoparticles, drugs, dyes and quantum dots for medical purposes [84]. The negative charge of nucleic acid is the key to the self-assembly of virus proteins to encapsulate nanoparticles [85, 86]. Programmed self-assembly is the latest trend of fabricating core-shell structure with nanoparticle and virus capsid for unique pharmaceutical

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applications [87, 88]. All these studies showed that the future of biological nanoparticle encapsulation for biomedical application with high biocompatibility and bioactivity is highly dependent on the development of intrinsic techniques for virus-based nanoparticle fabrication. 4. Biomedical application of virus-like nanoparticles

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Virus-like nanoparticles are utilized in biomedical application due to their significances such as enhanced biocompatibility, bioactivity and bioavailability in body fluids [89], nano-sized drugs carrying the potential to deliver them even by penetrating the cell’s nucleus and supreme self-assembly property [90]. Thus, virus-like nanoparticles are broadly subjected to biomedical applications such as targeted drug delivery [91], medical imaging [92], vaccines [59] and biosensors [93], since its time of discovery. 4.1. Targeted drug delivery

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The capsid of viruses act as protein cages in which drugs can be loaded in controlled and targeted delivery due to their ideal endocytosis size, well-defined geometry, less-toxic biodegradability and ability to functionalize at external, internal and inter-subunit interfaces via protein engineering [94, 95]. Self-assemblage of proteins lead to the formation of the caged protein capsid that are nano-sized and monodispersed capsules to accommodate drug molecules. The protein cargoes can be fabricated either

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with single or multiple protein as in pyruvate dehydrogenase E2 and CPMV, respectively [96, 97]. Also, protein engineering helps to control the surface charge, encapsulation of drugs, stability of particle and display of ligand in the viral capsid [98, 99]. Strategies such as protein engineering [100], chemical

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immobilization [101], non-covalent interactions [102] and structural changes through environmental triggering [102] are used for loading into and releasing drugs from viral capsid. In 2007, two orthogonal modification strategies were used to alter the exterior surface of genome-free MS2 viral capsid with polyethylene glycol (PEG) and 50-70 fluorescent dye copies were installed in the interior. The results revealed that the assemblage of capsid was not altered by covalent modification and the polymer coating helps to block 90% of the polyclonal antibody binding with the capsid surface proteins. Thus, the investigation showed that covalent modification of viral capsid allows self-assembly of proteins to form a viral drug cage such as the nano-cargo to deliver them at target site [103]. Likewise, self-assembled virus-like nanoparticles fabricated from the structural proteins VP6 of rotavirus was investigated for delivering an anticancer drug named doxorubicin (DOX). Initially, chemical conjugation was utilized to bind DOX with rotavirus capsid, which was later attached with VP6 via covalent modification to form a DOX-VP6 complex. The results showed that these virus-like nanoparticles based drug carriers are biocompatible, biodegradable and E. coli can be used for its

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ACCEPTED MANUSCRIPT scale-up production [104]. Also, protein cages of ferritin or apoferritin, small heat shock protein and viral capsid were used to synthesize drug compounds to deliver biomolecules such as RGD4C peptide, fluorescein isothiocyanate [105-107], doxorubicin [108], anti-CD4 antibody [109-111], antibody 19G2 [112] and poly-anethole sulphonic acid [113, 114], respectively. Recently, certain virus-like nanoparticles have started gaining importance in therapeutics and entered clinical trials as well as received approval from the United States Food and Drug Administration (USFDA) [115]. Systematic Mutation and Assembled Particle Selection (SyMAPS) technique is

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currently being explored to characterize the assembly competency of MS2 bacteriophage coat protein. The study revealed that the SyMAPS technique possesses the ability to fabricate self-assembled viral capsid to disassemble in late endosomes or early lysosomes which help in drug delivery applications [116]. Likewise, double arginine mutant of recombinant expressed tobacco mosaic virus coat protein (RR-TMV) was developed by self-assembled nanoscale disk. The RR-TMV was proved to be a

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promising protein-based nano-scaffold for targeted and controlled drug delivery [117]. The major drawback of using virus-like nanoparticles in drug delivery is the particle instability, intrinsic immunogenicity and limitations in antigen fusions. These challenges are addressed by using modified Hepatitis B core protein virus-like particles with enhanced functional properties by removing surface

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charge, artificial disulfide network introduction and new surface spike region transplantation [118]. Another study provided information on the presence of antimicrobials and cell-penetrating peptides in 272 capsid proteins derived from 133 viruses which includes both enveloped and non-enveloped virus. The main significance of this study is the ample proof that viruses are natural multivalent biotechnological platforms for drug delivery applications [119]. As mentioned before, there are certain drawbacks in utilizing virus-like nanoparticle for drug delivery [120] and extensive research to overcome these challenges will help to use them as a potential natural drug nanocarriers in future.

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4.2. Vaccines, medical imaging and biosensors

Vaccines for diseases caused by viruses are prepared by using dead viruses i.e. viruses without genetic material [121]. In recent times, virus-like nanoparticles are used to deliver vaccines, which plays a dual role as an immunity enhancer as well as vaccine delivery nano-cargoes. The proteins in the viral capsid help to trigger the production of antibody against the viral disease, whereas the vaccine inside the

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capsid helps to avoid any side effects or improve antigen-antibody interaction. Inhalation of in situ vaccination produced by using self-assembly of cowpea mosaic virus derived virus-like nanoparticles were proved to reduce B16F10 lung melanoma complications by the rapid generation of systemic

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antitumor immunity against poorly immunogenic B16F10 skin cells. Thus, these types of vaccines are proposed to be an excellent immunotherapeutic agent against metastatic cancer [77]. Likewise, viral capsid of Foot-and-mouth disease [122], polio [123], Crimean-congo hemorrhagic fever [124], Human Papilloma virus [125] and several other bioengineered virus-like nanoparticles [126] were used as vaccine and vaccine delivery cargoes to prevent the widespread of the diseases caused by these viruses. Further, certain virus-like nanoparticles were approved to be used as vaccines by USFDA such as Gardasil (Merck) and Cervarix (GlaxoSmithKline) against the Human Papilloma virus [127, 128]. Medical imaging is highly essential in diagnosing any disease [129] and the protein capsid of virus-like nanoparticle are recently introduced as nano-carriers to deliver imaging dyes at the target site [130]. Proteins of viral capsids act as contrast agents that can be useful in medical imaging applications such as magnetic resonance imaging (MRI) and positron emission tomography (PET) [131]. Numerous plant-based virus-like nanoparticles are also proved to be useful in intravital vascular imaging [132, 133] and MRI [134] by acting as a carrier for the delivery of fluorescent dyes [98] and MRI contrast

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ACCEPTED MANUSCRIPT agents including gadolinium [135] and divalent calcium ion [136, 137]. Similarly, viral capsid protein coated anisotropic gold nanorods are proved to be useful for background free imaging of living cell membrane with a dark field microscope [138]. Further, fluorescent nanodiamonds coated with the proteins of viral capsid are investigated and proved to be useful as glycosaminoglycans-specific cell imaging [139] where icosahedral shaped picornavirus capsid were patented to be useful in Nuclear Magnetic Resonance (NMR) and MRI [140]. Furthermore, viral capsids of physalis mottle virus [92] and several plant viruses such as CPMV, CCMV and potato virus X [141] were also used as

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nanocarriers to deliver imaging reagents, drugs and tissue specific imaging, respectively. Reduction in the complexity of fabrication, the chances of genotoxicity and mutation will enhance the usage of viral capsids for drug delivery and medical imaging application in future.

Virus-like nanoparticles are also utilized in fabricating biosensors. CPMV viral capsids were currently utilized to build a three-dimensional plasmonic nanostructure along with gold nanoparticles for

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real-time DNA detection [142]. Similarly, a recent report revealed an excellent interaction between non-functionalized gold nanoparticles and human papilloma (HPV16 L1) viral capsid, which will be useful for developing adsorption assays to detect HPV and prevent cervical cancer [143]. Also, tobacco mosaic virus (TMV) derived enzyme in nanotube morphology as nanocarriers were used as adapter

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templates. These viral capsids were utilized as amperometric glucose sensors for detecting glucose conjugates [144]. Further, virus-like nanoparticles from bacteriophages such as HK620 and HK97 showed enhanced detection of enteric E. coli bacteria in the range of 104 bacteria per ml in 1.5 hours [145]. Likewise, genetically engineered phage-templated manganese dioxide nanowires for glucose detection [146], TMV-based adapter templates [147], T7 bacteriophage induced morphology change in gold nanoparticles for sensitive plasmonic detection [148] and high sensitive MS2 viral capsid for nanoscale 129Xenon NMR detection [149] were some of the biosensor applications that are reported in

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recent times for biomolecule detection. Incorporation of specific receptors, antibodies and antimicrobial peptides on the surface of viral capsids will increase the utilization of virus-like nanoparticles for biosensor application in future. 4.3. Other novel applications

Virus-like nanoparticles are also used in applications such as enzyme delivery, antigen delivery and as

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protein supplements. Bacteriophage P22 virus-like nanoparticles were utilized for active encapsulation of enzymes [150, 151], a nano-vehicle for enzymatic activation of tamoxifen in tumor cells [152], T4 viral capsid for encapsulating active cyclic recombination recombinase and fluorescent mCherry

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plasmid DNA [153] and novel adeno-associated virus capsid were subjected to encapsulate enzymes [154]. These viral capsid encapsulated enzymes are highly significant in the treatment of enzyme deficiency and to deliver them in the target site. Likewise, antigen and antibodies were also encapsulated in virus-like nanoparticles for their enhanced targeted and controlled delivery [155-157]. Virosomes are nano-sized vesicles with an unilamellar phospholipid membrane that are produced by virus derived proteins [158]. In recent times, these virosomes are also used in vaccine [159] and drug delivery systems [72], which were also approved by USFDA in cases such as Epaxal, Inflexal V (Crucell) and Invivac (Solvay influenza) [160, 161]. Figure 2 is a schematic representation of various biomedical applications that are possible using viral-like nanoparticles.

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Figure 2: Schematics showing certain biomedical applications of virus-like nanoparticles 5. Gene therapy via virus nanoparticles – potential applications and challenges

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The void space covered by viral coat capsid protein, in the case of virus-like nanoparticles, are able to accommodate genes to deliver them on target site for gene therapy, similar to drug delivery [162]. Viral vectors are first hypothesized and developed into virus-based nanocarriers of genes to express beneficial proteins or to control cellular function [163]. The advantage of encapsulating therapeutic genes and delivering them through empty viral protein capsid is to avoid their enzymatic degradation in body fluids [164]. Several studies have been conducted to study the expression of antibodies after delivering genes through virus-like nanoparticles. In 2014, literatures suggested that engineered

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adeno-associated viruses possess the ability to accommodate and deliver genes for clinical gene therapy [7]. Hence, protein capsids of adeno-associated viruses were recently utilized for the encapsulation and delivery of broadly neutralizing antibodies (bNAbs) to inhibit the growth of human immunodeficiency virus (HIV) as method for HIV prevention [165]. Intravitreal adeno-associated viral vectors faces an obstacle in antibody neutralization while delivering gene, when investigated in non-human primate

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models [166]. Numerous adeno-associated virus based products as gene delivery vehicle systems were fabricated later, which were approved by USFDA (Luxturna) and in Europe (Glybera) [167]. Human clinical trials were successful for recombinant adeno-associated virus based gene delivery system due

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to 20 years of intense research which leads to the development of second generation gene delivery vectors enhancing host antibody response for improving immunity in patients [168]. Likewise, the chimeric viral capsid protein of hepatitis B virus along with p19 RNA binding protein and integrin-binding peptide was fabricated and proved to be highly useful for the delivery of small interfering RNA (siRNA) [169]. Similarly, empty protein capsids of hepatitis B and various plant viruses were reported to be used to accommodate genes for therapeutic applications [137, 170]. All these studies showed that virus-like nanoparticles are extensively used in gene therapy application, in spite of the challenges such as unexpected mutation and genotoxicity. 6. Proposed mechanism of virus nanoparticle-based gene delivery A rapid growth in the field of gene therapy using virus-like nanoparticles leads to the existence of several literatures that bothers on the fabrication of virus-like nanoparticles and encapsulating therapeutic genes in them. However, there exists a research gap in explaining the mechanism of virus-like nanoparticles in delivering genes in the target site. Thus, a possible mechanism was proposed

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ACCEPTED MANUSCRIPT in this review for virus-like nanoparticle mediated targeted gene delivery as shown in Figure 3. Initially, empty viral capsid has to be prepared to form virus-like nanoparticles by removing their genetic material through genetic engineering [171]. Later, therapeutic gene has to be loaded into the empty viral protein capsid of virus-like nanoparticles via infusion or chemical conjugation [172]. The therapeutic gene in virus-like nanoparticle was subjected to molecular conjugation either with nanoparticles, polymers or biomolecules to increase their cellular compatibility and binding efficiency towards host cell receptor [173, 174]. Upon binding over the host cell receptor on the cell surface, the

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conjugated molecules degrade to allow the virus-like nanoparticle to bypass the cell membrane and enter into the cell. The proteins in the viral capsid are either degraded by the cytoplasmic enzymes or binds with the cellular nucleus to release the therapeutic gene. The molecular conjugation plays a critical role which enables the virus-like nanoparticles to deliver therapeutic genes in the target site [175]. Similarly, the therapeutic gene sequence is bound with the viral genome of virus nanoparticle

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which can deliver them in the target site [98]. The current and future research focuses on programming virus-like nanoparticles to produce more therapeutic genes in the target cells by self-transcription and translation process to cure genetic diseases which will be a breakthrough in gene therapy. However, challenges such as unexpected mutation, cytotoxicity and genotoxicity have to be taken into

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consideration, while using novel virus-based nanostructures for clinical and pre-clinical analysis.

Figure 3: Schematic showing possible mechanism of virus-like nanoparticles in gene delivery 7. Conclusion

The primary aim of this review article is to provide information on the recent developments in virus-like nanoparticles for biomedical applications. It is observed that the developments in genetic engineering lead to the increased fabrication of numerous virus-like nanoparticles for biomedical applications such as drug, vaccine and antigen delivery, medical imaging and biosensors. Due to the successful incorporation of virus-like nanostructures in these biomedical applications, it was introduced in gene therapy in recent times. However, there is very little literature available to support the widespread application of virus-like nanoparticles in gene therapy and there exists a vague clarity in the mechanism of gene delivery procedures through viruses. Thus, the current review will provide an insight into the mechanism of delivering gene which is possible through viruses. In future, programmed

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ACCEPTED MANUSCRIPT virus-like nanostructures are key in the development of self-healing gene therapy tools which will act as nanosized robots to alter and deliver genes to cure genetic diseases. 8. Reference [1] T. Strachan, A.P. Read, Human molecular genetics, Chromosome Res. 4 (1996) 475-475. [2] L. Naldini, Gene therapy returns to centre stage, Nature 526 (2015) 351-360. [3] S.A. Rosenberg, P. Aebersold, K. Cornetta, A. Kasid, R.A. Morgan, R. Moen, E.M. Karson, M.T.

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Highlights •

The manuscript focuses on highlighting the possibilities of using virus-like nanoparticles for targeted gene delivery.

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The virus-like nanoparticles are proposed in the current review to be a novel

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gene therapy vector in future for delivering therapeutic genes in the target site. Additionally, other biomedical applications that are explored using virus-like nanoparticles and their probable mechanism to deliver genes were also

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discussed.